Cell Death and Differentiation (2003) 10, 1005–1015 & 2003 Nature Publishing Group All rights reserved 1350-9047/03 $25.00 www.nature.com/cdd Caspase-9 is activated in a cytochrome c-independent manner early during TNFa-induced apoptosis in murine cells

MA McDonnell1, D Wang1, SM Khan1, MG Vander Heiden2 and Introduction A Kelekar*,1,3 Apoptosis is a tightly regulated process important for differentiation, for the regulation of cell numbers, and for the removal of aged, damaged and autoreactive cells.1,2 1 Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis MN 55455, USA; A variety of extracellular and intracellular signals can trigger 2 Pritzker School of Medicine, University of Chicago, Chicago, IL 60637, USA; an apoptotic response, including growth factor deprivation, Present address: Brigham and Women’s Hospital, Department of Medicine, overexpression of oncogenes and tumor suppressor genes, Boston, MA 02115 radiation, chemotherapeutic drugs, and crosslinking of 3 Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA receptors such as the Fas or tumor necrosis factor (TNF) * Corresponding author: A Kelekar. Tel: 612-625-3204; Fax: 612-625-1121; receptor. Apoptotic triggers activate intracellular response E-mail: [email protected] pathways that lead to the controlled activation of cysteine proteases known as caspases.3 Two major pathways Abstract of caspase activation have been described – one involving the release of multiple polypeptides from mitochondria as a FL5.12 pro-B lymphoma cells utilize the mitochondrial pathway result of its destabilization and the other involving cell surface to apoptosis in response to tumor necrosis factor (TNF) ‘death’ receptor activation by ligand binding.4 receptor occupation, yet high levels of the Bcl-2 family Human cells have been classified as type I or type II based antiapoptotic protein, Bcl-xL, fail to protect these cells against on their responsiveness to activation of the TNF family of 5 TNF-receptor-activated death. Bcl-xL expression delays, but death receptors, particularly Fas ligand-mediated death. does not totally block, the release of mitochondrial cytochrome Ligand binding causes trimerization of these receptors on the c (cyt c) in these cells in response to TNFa-induced apoptosis cell surface and recruitment of cytoplasmic adaptor proteins. and caspase-9 is processed prior to mitochondrial cyt c release These, in turn, recruit procaspase-8 molecules that self- process into their active forms.6,7 Once activated, caspase-8 under these circumstances. Early processing of caspase-9 can trigger two distinct pathways of apoptosis. In type I cells, also occurred in Apaf-1 knockout murine fibroblasts in the release of large amounts of activated caspase-8 from the response to TNF-receptor occupation. A caspase-9-specific death-inducing signaling complex (DISC), enables direct inhibitor was more effective in delaying the progression of activation of downstream caspases such as caspase-3, apoptosis in the FL5.12 Bcl-xL cells than was an inhibitor leading to cell death. In contrast, in type II cells DISC specific to caspase-3. Furthermore, downregulation of cas- formation is greatly reduced, but the small amounts of active pase-9 levels by RNA interference resulted in partial protection caspase-8 molecules that are generated are sufficient to of these cells against TNF-receptor-activated apoptosis, induce mitochondrial apoptogenic activity. Caspase-8 indicating that caspase-9 activation contributed to early cleaves the Bcl-2 family protein, Bid, to generate a C-terminal amplification of the caspase cascade. Consistent with this, fragment that then translocates to the mitochondria causing proteolytic processing of caspase-9 was observed prior to its disruption, and triggering the release of cytochrome c (cyt c).8-10 A cytoplasmic multiprotein complex, comprising Apaf- processing by caspase-3, suggesting that caspase-3 was not 1, cyt c and the ‘initiator’ caspase, caspase-9, then activates a responsible for early caspase-9 activation. We show that series of ‘effector’ caspases beginning with caspase-3 and murine caspase-9 is efficiently processed by active caspase-8 culminating in the death of the cell.11,12 In cells of type II, such at SEPD, the motif at which caspase-9 autoprocesses following as Jurkat, the proapoptotic effects of activated Bid on the its recruitment to the apoptosome. Our results suggest that, in mitochondria can be effectively inhibited in the presence of 5,13 addition to processing procaspase-3 and the BH3 protein Bid, high levels of Bcl-2 or Bcl-xL proteins. Murine hepatocytes active caspase-8 can cleave and activate procaspase-9 in appear to exhibit properties of type II cells,14 but some response to TNF receptor crosslinking in murine cells. hematopoietic cell lines of murine origin have eluded Cell Death and Differentiation (2003) 10, 1005–1015. doi:10.1038/ categorization as type I or type II in terms of their respon- sj.cdd.4401271 siveness to Fas and/or TNF-receptor activation. For instance, IL-3-dependent murine pro-B FL5.12 cells recruit small Keywords: apoptosis; caspase-9; caspase-8; cytochrome c amounts of procaspase-8 to the DISC and clearly utilize Bid release; death receptors cleavage as the primary mechanism of amplification15 (AK, unpublished). Despite this, however, overexpressed Bcl-xL Abbreviations: TNF-a, tumor necrosis factor-alpha; CHX, cy- cannot protect FL5.12 cells against TNF-receptor-mediated cloheximide; cyt c, cytochrome c; DISC, death-inducing signaling death. It was of interest, therefore, to understand how complex amplification of the apoptotic process occurred in these and Activation of caspase-9 by caspase-8 MA McDonnell et al 1006

other murine cell types in response to TNF-receptor cross- a 100 linking. Release of mitochondrial cyt c into the cytoplasm and its 80 subsequent association with the Apaf-1 protein is thought to Neo be an absolute requirement for the activation of caspase-9, 60 Bcl-x the apical caspase in the mitochondrial pathway of apopto- L sis.12 In the presence of ATP/dATP, cyt c that has been 40 released into the cytosol binds to and triggers the oligomer- ization of the cytosolic Apaf-1 protein. The resultant complex % viability recruits multiple copies of procaspase-9 leading to its 20 activation.12 Although autoprocessing occurs rapidly, unpro- 0 cessed forms of caspase-9 are also catalytically active as part 0 5 10 15 20 25 16,17 of the caspase-9–Apaf-1 holoenzyme complex. A purified Hours following IL3 withdrawal truncated form of the Apaf-1 protein lacking the WD-repeats and cyt c binding site was shown in vitro to be constitutively 100 active in its ability to induce self-processing of procaspase- b 18 9. However, two recent studies have indicated that caspase- 90

9 can also be activated by mechanisms involving neither cyt c Neo TNF- /CHX 19,20 80 release nor Apaf-1 activation. One study showed that, in Neo TNF- /CHX + z-VAD response to endoplasmic reticular (ER) stress, caspase-12 70 Bcl-x L TNF- /CHX was able to process and activate caspase-9, while another 60 Bcl-x L TNF- /CHX + z-VAD indicated that virus infection could trigger a novel, yet 50 unknown, pathway of caspase-9 cleavage. % viability

The primary objective of the present study was to determine 40 the basis for the inability of overexpressed Bcl-xL to protect 30 FL5.12 pro-B cells from launching a rapid apoptotic response 0 51015 to TNF-a/cycloheximide (CHX) treatment, when the mito- Hours following treatment with TNF- /CHX chondrial pathway (via Bid cleavage) and cyt c release Figure 1 Bcl-xL protects murine FL5.12 pro-B cells against growth factor appeared to be the preferred route to apoptotic death in withdrawal, not against death receptor activation. FL5.12 cells transfected with untransfected and vector-transfected controls. either pSFFV–Bcl-xL or with the empty vector, pSFFV–Neo, were cultured in IL-3- free medium for 22 h (a) or incubated with TNF-a (5 ng/ml) and CHX (20 mg/ml) in the presence or absence of 50 mM of the caspase inhibitor z-VAD-fmk for 12 h Results (b). Viability (mean and standard deviations, n¼3) of the transfected clones under conditions of IL3 deprivation was determined by PI exclusion and FACS Bcl-xL does not protect murine pro-B cells from analysis at the indicated time points TNFa-induced death Interleukin-3 (IL-3)-dependent murine FL5.12 pro-B lympho- cells exposed to TNF-a/CHX for a few hours. The Western blot ma cells expressing high levels of transfected Bcl-xL or the in Figure 2a (top panel) shows that 3 h after induction, when empty Neo vector were either subjected to a growth factor apoptotic cells are easily detectable by flow cytometry withdrawal assay or treated with TNF-a in the presence of (Figure 1b), mitochondria from Bcl-xL-expressing FL5.12 cells CHX (described in Materials and Methods). Figure 1a and b still retain cyt c. Shown in the center and bottom panels are shows that FL5.12 cells overexpressing Bcl-xL are protected cytochrome oxidase subunit IV (COX IV) and actin Western against growth factor withdrawal-induced death, as assayed blots, controls for the mitochondrial and cytosolic fractions, by propidium iodide (PI) exclusion and flow cytometry, for as respectively. Bcl-xL protein levels, as detected by Western long as 96 h (22-h experiment shown in Figure 1a), but not blotting, did not decrease for at least 4 h following treatment against apoptosis caused by treatment with TNF-a and CHX. with either TNF-a/CHX or CHX alone (not shown). The cyt c However, TNF-a/CHX-induced death in both control and Bcl- release observed in the FL5.12 Neo cells is the result of xL cells could be inhibited in the presence of a broad-spectrum caspase-8-mediated cleavage and activation of the Bcl-2 caspase inhibitor, z-VAD-fmk. The inhibition in control cells family BH3 protein, Bid, in response to TNF-receptor cross- 15 was manifest as a delayed death response, while the Bcl-xL linking. We were able to confirm the cleavage of Bid in death cells exhibited almost total resistance to apoptosis for at least receptor-activated control and Bcl-xL-expressing cells by 24 h (a 12-h experiment is shown in Figure 1b). Western blotting using antibodies against both full-length and cleaved mouse Bid protein (not shown). However, while Johnson et al.15 demonstrate no release of cyt c from Bcl-xL delays the release of mitochondrial cyt c in mitochondria of FL5.12 Bcl-xL cells as late as 6 h after response to TNF-receptor activation induction of apoptosis, our data consistently show 30–50% Since Bcl-xL has been shown to be effective at maintaining loss of the protein from mitochondria in Bcl-xL cells by this time mitochondrial integrity and preventing cyt c release during point and almost total release 12 h post-treatment. The data growth factor withdrawal,21 we determined the cellular are summarized in Figure 2b. It may be noted that the Neo- distribution of cyt c in control and Bcl-xL-expressing FL5.12 and Bcl-xL-expressing lines used in our experiments were

Cell Death and Differentiation Activation of caspase-9 by caspase-8 MA McDonnell et al 1007

clonal isolates. Additionally, the Bcl-xL lines were selected for cyt c-dependent caspase-9 pathway in the amplification of high levels of expression of the transfected plasmid.22 Under downstream apoptotic cascades in response to TNF-a, it was conditions of IL-3 withdrawal, these cell lines show mitochon- important to determine the mechanism underlying the early drial retention of cyt c for at least 72 h postdeprivation (not and rapid apoptosis of the Bcl-xL-expressing cells in the shown). Since FL5.12 control cells primarily adopt the (type II) absence of cyt c release.

Caspase-9 is processed prior to mitochondrial cyt a untreated TNF- /CHX c release in response to TNF-receptor occupation

NeoBcl-xL Neo Bcl-xL in Bcl-xL-expressing FL5.12 cells M S MMMSSS Next, we examined the processing of caspase-9 and -3, the cyt. c two major proteases associated with mitochondrial and nonmitochondrial pathways downstream of activated cas- pase-8, under conditions of TNF-receptor crosslinking. COX IV Figure 3 shows lysates of FL5.12 control and Bcl-xL cells at 0, 2, 3, 4 and 6 h after treatment with TNF-a/CHX, immunoblotted with antibodies against caspase-9 (upper actin panels) or a cleaved (active) form of caspase-3 (lower panels). Caspase-9 is being processed within the first 2 h of induction in both the cell lines. We have consistently observed

b 100 the early cleavage of caspase-9 in response to TNF-a; the results shown in Figure 3 are representative of six different experiments. A number of groups have already established

75 that caspase-9 requires cyt c release and recruitment into the apoptosome to autoprocess and become activated.12,23

Neo However, data in Figure 2 showing effective inhibition of cyt 50 c release by overexpressed Bcl-xL for three or more hours Bcl-xL after induction of apoptosis through the TNF receptor (Figure 2), taken together with results in Figure 3, strongly 25 indicate that caspase-9 is processed prior to mitochondrial cyt c release in the FL5.12 cells. The cleaved caspase-3 (Figure 3, Percent cytochrome c in cytosol lower panels) detected within the first 3 h after induction 0 probably results from caspase-8 processing activity. It may be 02 34612 Hours following treatment with TNF- α/CHX noted that levels of this cleaved, presumably active, form increase noticeably at later time points in both vector and Bcl- Figure 2 Bcl-xL prevents early mitochondrial cytochrome c release in FL5.12 x -expressing cells, and this was confirmed using a colori- cells in response to TNF-a/CHX treatment. (a) Western blot of fractionated L metric caspase-3 activity detection kit (R&D Systems, not FL5.12 cells showing the distribution of cyt c. Untreated Neo and Bcl-xL FL5.12 cells, and cells treated with TNF-a/CHX for 3 h were fractionated into shown). Additionally, it may be noted that the 38 kDa mitochondrial (M) and cytoplasmic, S100 (S), fractions, and immunoblotted with processed product of caspase-9 consistently appeared at a monoclonal antibody against cyt c, COX IV and actin (see Materials and least as early after TNF treatment as the 40 kDa cleavage Methods). (b) Bar graph showing the ratio of cytosolic cyt c to total cyt c in Neo product expected from caspase-3 processing activity and Bcl-xL FL5.12 cells 0, 2, 3, 4, 6 and 12 h following apoptotic induction with TNF-a/CHX (mean and standard deviations, n¼3). Fractionated cell extracts (Figure 3, upper panels). It was important to determine were immunoblotted with antibody against cyt c and the chemiluminescent cyt c whether the observed early processing of caspase-9 was bands, visualized by autoradiography, were later quantified by densitometry leading to its activation or was merely a consequence of the using a BioRadGS-363 Molecular Imager degradative activity associated with apoptotic progression.

hours following 0234 6 02 34 6 TNFα/CHX addition 50 kD Caspase-9 40 kD 38 kD

Active 17 kD Caspase-3

Neo Bcl-xL

Figure 3 Caspase-9 processing in FL5.12 Bcl-xL cells occurs within 2 h of apoptotic induction with TNF-a/CHX. Autoradiographs of lysates from Neo and Bcl-xL FL5.12 cells treated with TNF-a/CHX for 0, 2, 3, 4, 6 and 12 h immunoblotted with antibody against caspase-9 (top panels) or a cleaved form of caspase-3 (bottom panels)

Cell Death and Differentiation Activation of caspase-9 by caspase-8 MA McDonnell et al 1008

Early caspase-9 activity contributes to the rapid a 100 100 amplification of apoptosis in Bcl-xL-expressing FL5.12 cells in response to TNF-a 75 75 We predicted that if early caspase-9 activation was critical for the rapid and early amplification of this apoptotic pathway, 50 50

inhibiting caspase-9 activity would delay apoptosis. To test % viability % viability this, FL5.12 cells were treated with TNF-a and CHX in the 25 25 presence of either the pan-caspase inhibitor z-VAD-fmk, or specific caspase inhibitors. Shown in Figure 4a are bar graphs 0 0 depicting the viability of FL5.12 cells at 6 h following TNF-a +IETD +IETD +LEHD +LEHD +z-VAD +z-VAD +DQMD +DQMD /CHX 6hr treatment, as assayed by flow cytometric analysis of Annexin /CHX 6hr untreated untreated α α V and PI uptake. As expected, z-VAD-fmk blocked apoptosis TNF-α/CHX TNF-α/CHX TNF- TNF- of the Bcl-xL cells while providing only partial protection to FL5.12 Neo FL5.12 Bcl-xL the controls. It has previously been shown that 50 mM z-VAD- fmk is unable to block cyt c release in FL5.12 Neo cells15 b (and AK, unpublished). The caspase-3-specific inhibitor, DQMD IETD LEHD ___ z-DQMD-fmk, imparted 30–50% protection over the TNF-a/ inhibitor + +++++ TNF−α/CHX _ +++ +++++ CHX controls in both the cell lines. It is known that related Time in hr. 036363636 effector caspases, such as caspase-7, can effectively replace active caspase-324,25 in processing downstream caspase -9 targets, and this may be the reason for the lower levels of protection observed in the presence of z-DQMD-fmk. The caspase-8 inhibitor, z-IETD-fmk, was more effective in 1 2 3 4 5 6 7 8 9 inhibiting apoptosis in both the cell lines. Figure 4a also cleaved shows that z-LEHD-fmk, an inhibitor of caspase-9 activity, caspase-3 delayed apoptosis almost as efficiently in the Bcl-xL cells as did z-IETD (right panel), suggesting that caspase-9 proteolytic c activity plays an important role in this death pathway even in _ + + + TNF-α/CHX the absence of cyt c release. The low level of inhibition 0366Time in hr. _ _ _ observed in control FL5.12 cells (Figure 4a, left panel) in the + z-VAD-fmk presence of the caspase-9 inhibitor can be attributed to the caspase-9 other proapoptotic compounds, such as Smac/DIABLO, AIF, cleaved and even preprocessed caspase-3, that are released from the caspase-3 mitochondrial intermembrane space along with cyt c. actin Although, IETD and LEHD were able to delay the onset of apoptosis more effectively than the caspase-3 inhibitor, only Figure 4 Early caspase-9 processing contributes to the rapid amplification of the broad-spectrum inhibitor z-VAD could protect the Bcl-xL apoptosis observed in Bcl-xL-expressing FL5.12 cells in response to TNF-a/CHX. cells against TNF-induced death for over 24 h (not shown). (a) FL5.12 cells were incubated with 100 mM concentrations of specific cell- Figure 4b is a Western blot of FL5.12 Bcl-x cell lysates permeable caspase inhibitors z-DQMD-fmk (caspase-3), z-IETD-fmk (caspase- L 8) or z-LEHD-fmk (caspase-9) and 50 mM of the pancaspase inhibitor z-VAD-fmk showing caspase-9 and -3 processing, 3 and 6 h following for 30 min before being treated with TNF-a and CHX. Viability of cells was TNF-a/CHX treatment in the presence or absence of caspase determined at specific intervals by flow cytometric analysis Annexin V–FITC inhibitors. While the caspase-8 inhibitor, IETD, effectively labeling and PI uptake (see Materials and Methods). Figure represents percent prevented processing of both caspase-9 and -3 (lanes 6 and viability of untreated FL5.12 Neo (left panel) and Bcl-xL (right panel) cells, and 7), the caspase-3 inhibitor (lanes 4 and 5) did not affect the cells treated with TNF-a/CHX for 6 h in the absence or presence of caspase inhibitors (mean and standard deviations, n¼3). (b) Western blots of lysates from processing of either of these caspases. Figure 4c (top panel) 0, 3 and 6 h-treated FL5.12 Bcl-xL cells from the experiment in (4a). Top panel represents a caspase-9 immunoblot of lyates from untreated shows an autoradiograph of 25 mg total protein immunoblotted with antibody FL5.12 Bcl-xL cells, and cells treated with TNF-a/CHX in the against caspase-9, and bottom panel represents the same Western blot, stripped presence of 50 mM z-VAD. The membrane was sequentially and reprobed with an antibody against the cleaved (active) form of caspase-3. (c) Caspase-9 immunoblot of z-VAD-fmk-incubated Bcl-xL cell lysates (top panel) stripped and reprobed with antibodies against active caspase- from the above experiment, stripped and reprobed with active caspase-3 (center 3 and actin (center and bottom panels, respectively). As panel) and actin (bottom panel) antibodies expected, based on the viability assays shown in Figure 4a (right panel), both caspase-9 and -3 processing were of LEHD added 30 min before or after the addition of inducer completely inhibited. (Figure 5a). Results indicated that caspase-3 activity was Although caspase-9 inhibition effectively blocked the effectively blocked at the earliest time point in both the processing of caspase-3 at early times, by the end of 6 h instances. Activity remained low in both samples at the end of levels of cleaved caspase-3 appeared to have been restored 6 h, and consistently showed steady increases in DEVDase to those observed in the controls (Figure 4b, lower panel, levels thereafter (not shown). If caspase-3 was being directly lanes 8 and 9). To resolve this issue, DEVDase activity was inhibited by LEHD rather than through inhibition of caspase-9, measured in TNF-treated FL5.12 Bcl-xL cells in the presence we should also detect caspase-8 inhibition, since caspase-8

Cell Death and Differentiation Activation of caspase-9 by caspase-8 MA McDonnell et al 1009 a 0.6 Bcl-xL TNF- /CHX Bcl-xL TNF- /CHX + LEHD (30 min. prior) 0.4 Bcl-xL TNF- /CHX + LEHD (30 min. after)

IETDase activity 0.2 Time after (arbitrary units) addition of

DEVDase activity (arbitrary OD units) TNF-α/CHX - LEHD + LEHD

0 hr 0.04 (+ 0.008) 0.035 (+ 0.006) 0 3 hr -2 0 3 6 0.24 (+ 0.05) 0.18 (+ 0.03) Hours following treatment with TNF-α/CHX b

0.6 Bcl-xL TNF- /CHX

0.5 Bcl-xL TNF- /CHX + DQMD (30 min. prior) Bcl-x TNF- /CHX + DQMD (30 min. after) 0.4 L

0.3

0.2 LEHDase activity (arbitrary OD units) 0.1

0 -2 0 3 6 Hours following treatment with TNF-α/CHX Figure 6 Downregulation of caspase-9 levels by RNA interference partially protects FL5.12 Bcl-xL cells against TNF-receptor-activated apoptosis. FL5.12 Figure 5 Inhibition of caspase-9 inhibits early DEVDase activity, but inhibition Bcl-xL cells were either mock transfected or transfected with mC-9 siRNA of caspase-3 does not affect early LEHDase activity in FL5.12 Bcl-xL cells treated (100 nM) using siPORT Amine (Ambion) as transfection agent. (a) Western blot with TNF-a/CHX. Cells were incubated with 100 mM z-LEHD-fmk (a) or z-DQMD- of lysates from parental FL5.12 Bcl-xL cells (lane 1), mock-transfected FL5.12 fmk (b) 30 min prior to, or 30 min. following (shown by arrows) the addition of Bcl-xL cells (lane 2) and cells transfected with caspase-9 siRNA (lane 3). Upper TNF-a/CHX, and DEVDase (a) or LEHDase (b) activity measured at the panel represents an immunoblot of caspase-9 and lower panel shows the same indicated time points using colorimetric assays (R&D Systems). Table inset in (a) blot stripped and reprobed with an antibody against the Bcl-xL protein. shows IETDase activity measured at 0 and 3 h in the FL5.12 cells treated with z- (b) Untransfected, mock-transfected and caspase-9 siRNA-transfected FL5.12 LEHD-fmk prior to apoptosis induction Bcl-xL cells were treated with TNF-a and CHX and viability of cells was determined by flow cytometric analysis of Annexin V–FITC labeling and PI uptake. Bar graphs represent percent viability of unstimulated and 6 h-stimulated and -9 are more closely related in terms of their substrate cell populations (n¼3) specificities.26 However, IETDase activity measured at 0 and 3 h in the FL5.12 cells treated with LEHD prior to apoptosis induction, indicated that caspase-8 was not significantly inhibited at the 3 h time point (Table inset, Figure 5a). The xL cells, it was important to confirm this by methods that 25% reduction observed in IETDase activity is probably did not involve the use of synthetic caspase inhibitors. For responsible for part of the decrease in caspase-3 activity, but an alternative approach, FL5.12 Bcl-xL cells were transiently we believe that these data indicate that a bulk of the early transfected with siRNA directed against nucleotides 289–309 inhibition of caspase-3 activity results from inhibition of of the murine caspase-9 (mC-9) RNA sequence (see caspase-9 function. Materials and Methods). Figure 6a shows a caspase-9 Western blots of caspase-9 in Figure 4b (upper panel, lanes immunoblot of lysates 48 h following transfection 4 and 5) showed no effect of DQMD, the caspase-3-specific from parental, mock transfected and siRNA-transfected synthetic inhibitor, on caspase-9 processing. Colorimetric FL5.12 Bcl-xL cells (upper panel). The membrane was caspase-9 activity assays in the presence and absence of stripped and reprobed with an antibody against Bcl-xL to DQMD (Figure 5b) showed that caspase-3 inhibition did not confirm even loading of lysates (lower panel). The decrease in block early caspase-9 activation, but inhibited late LEHDase caspase-9 protein expression was determined by densito- activity. These results confirm that caspase-9 is activated metry to be roughly 50%. Figure 6b shows the results of within 3 h following TNF-receptor crosslinking and suggest, viability assays carried out on control and transfected furthermore, that active caspase-3 is not the protease populations treated with TNF-a and CHX for 6 h. While the responsible for the early activation. mean viability exhibited by the mock-transfected cells did not differ from the 48% viability observed in the untransfected parental population, caspase-9 siRNA-transfected cells were RNA interference-mediated downregulation of 64% viable at the end of 6 h. The observed delay in apoptosis in the presence of reduced levels of caspase-9 supports endogenous caspase-9 protects FL5.12. Bcl-x L earlier results (see Figures 4 and 5) that caspase-9 function is cells from TNF-receptor-activated death involved in accelerating TNF-activated apoptosis in cells in Although data in Figure 4 were suggestive of caspase-9 which the mitochondrial cyt c release pathway is effectively participation in early amplification of apoptosis in FL5.12 Bcl- blocked.

Cell Death and Differentiation Activation of caspase-9 by caspase-8 MA McDonnell et al 1010

Early caspase-9 processing occurs in cells lacking Murine caspase-9 is processed by active caspase- the Apaf-1 protein 8inanin vitro cleavage assay Having determined that the early proteolytic activity of Yet another possibility was that active caspase-8 molecules caspase-9 was playing a role in the amplification of apoptosis were themselves directly acting on procaspase-9 to process in the Bcl-xL-transfected cells, we proceeded to determine the and activate it. Although caspase-8 had been shown to source of this early activation. It was unlikely that active activate caspase-9 indirectly in type II cells via cleavage of the caspase-3 molecules resulting from the proteolytic activity of Bid protein and the subsequent release of mitochondrial cyt c, caspase-8 were acting on procaspase-9 to process and to date there has been no evidence of direct processing of activate it (Figures 4 and 5). It was possible that a caspase-9 in vivo by this initiator caspase. constitutively active form of Apaf-1 was being generated in The autoprocessed form of caspase-9 is 38 kDa (see response to receptor occupation that could bypass the Figure 3, upper panels), but autoprocessing of the latter has requirement for cyt c association in order to activate the only been shown to occur in vivo by ‘induced proximity’ in the autoprocessing of procaspase-9 molecules. We surmised that context of apoptosome formation in reponse to cyt c if Apaf-1 was required for the early activation of caspase-9 in release.23,27 The autoprocessing motif, SEPD (residues the TNF-a pathway, then early processing of caspase-9 350–353), in mC-9 is preceded directly by LDSD (346–349), should not be detectable in Apaf-1-deficient cells exposed to both putative caspase-8 cleavage consensi26 (see Figure 8a). TNF. Additionally, by using Apaf-1À/À cells, we would ensure The LDSD motif is absent in the human caspase-9 (hC-9) that no activation of caspase-9 could occur via the cyt c protein. release pathway, while eliminating the need for overexpres- Full-length murine procaspase-9 was cloned from mouse sing Bcl-xL to block this pathway. Figure 7 shows Western brain cDNA and tagged with a myc epitope at the C-terminus. blots of lysates from ApafÀ/À and control mouse embryo Two point mutants of murine caspase-9, LDSA and SEPA, fibroblasts (MEFs) that had been treated with TNF-a/CHX in were also generated in which residues D349 and D353, the presence or absence of z-VAD-fmk, the broad-spectrum respectively, were replaced with alanine. Radiolabeled in caspase inhibitor. Upper panel in Figure 7 shows that vitro translated murine caspase-9, LDSA and SEPA, as well caspase-9 is processed in both types of MEFs within 3 h as in vitro translated human wild-type protein were incubated following exposure to TNF-a and CHX. Although, z-VAD either with active recombinant caspase-3 or -8 at 371C for 1 h. completely inhibits this processing in Apaf-1À/À cells, control Figure 8b shows an autoradiograph of SDS-PAGE analysis of MEFs continue to activate caspase-9 at least partially via the the reactions. Active caspase-3 (lanes 5–8) processed both mitochondrial pathway. The Western blot was stripped and the human and murine proteins generating cleavage products reprobed with an antibody against Apaf-1 (Figure 7, lower of the expected size; 40 kDa for murine caspase-9 and 37 kDa panel). These data suggest that the early cyt c-independent for the human protein. The panel on the right (lanes 9–12) processing of caspase-9 in the TNF pathway in these cells did shows the results of active caspase-8 processing activity on not result from Apaf-1 activity. caspase-9 and the two point mutants. A major processed fragment of 38 kDa and a minor 40 kDa product resulted from cleavage of wild-type mC-9 as well as the LDSA mutant. The 38 kDa fragment generated by caspase-8 cleavage activity is Apaf-1+/+ Apaf-1-/- absent in the cleavage reaction with the SEPA mutant (lane _ _ TNF-α/CHX ++ + + 11) indicating that it was the SEPD, and not the LDSD, motif __ __ that served as a cleavage site for active caspase-8. Two z-VAD + + cleavage products of approximately 37 and 35 kDa, resulting from processing at the DQLD and PEPD motifs, were also observed in the cleavage reaction with human caspase-9 (lane 12), however, processing at PEPD by active caspase-8 Caspase -9 was consistently weaker than at the corresponding (SEPD) motif in the murine protein (lane 9). Srinivasula et al.28 have demonstrated the cleavage of purified recombinant hC-9 by active caspase-8, and their data indicate that although hC-9 is processed at both sites, the DQLD motif is the preferred site of cleavage. Thus, mC-9 serves as a direct processing substrate Apaf-1 for active caspase-8, and may be one of the first caspases to be activated in the TNF pathway of apoptosis.

Discussion Figure 7 Early caspase-9 processing in murine fibroblasts following exposure to TNF-a/CHX is dependent on caspase activity and not on the Apaf-1 protein. Human cells have been classified as cells of type I or type II The figure shows autoradiographs of Western blots from lysates of Apaf þ / þ and À/À based on the nature of their response to death receptor Apaf MEFs that had been treated with TNF-a/CHX for 3 h in the presence or 5 absence of the caspase inhibitor z-VAD-fmk. Upper panel shows caspase-9 activation by ligands such as Fas and TNF-a. In cells of type I processing, and lower panel shows the same Western blot, stripped and death receptor ligation leads to vigorous activation of reprobed with an antibody against Apaf-1 (see Materials and Methods) caspase-8, followed by the direct cleavage and activation of

Cell Death and Differentiation Activation of caspase-9 by caspase-8 MA McDonnell et al 1011

a caspase-3 murine caspase-9 auto-processing cleavage site ? site 323 QACGGEQKDHGFEVACTSSQGRTL* DSDSEPDAVPYQEGPRPL DQL DA QACGGEQKDHGFEVAS TSPEDESPGS NPEPDAT P F QEGL R TFDQL DA 285 * auto-processing caspase-3 human caspase-9 site cleavage site

b control active casp-3 active casp-8 mc-9 + + + LDSA + + + SEPA + + + hc-9 + + + 1 2 3 4 5 6 7 8 9 10 11 12

49.8 kD

40 kD 38 kD 35.8 kD

Figure 8 Active recombinant caspase-8 cleaves murine caspase-9 at SEPD, the site of caspase-9 autoprocessing. (a) Representation of the amino-acid sequence between the active site, QACGG (*), and the caspase-3 processing site (marked by arrows) in mC-9 and hC-9. Conserved residues are shaded in black. Additional arrows depict the caspase-9 autoprocessing sites, SEPD (mouse), PEPD (human) and a putative caspase-8 cleavage consensus motif, LDSD (?) present only in the murine protein. (b) In vitro synthesized, radiolabeled mC-9, LDSA, SEPA or hC-9 proteins were synthesized using the TNT transcription/translation system (Promega) and incubated with 15 mU/ml of active human recombinant caspase-3 (lanes 5–8), or 150 mU/ml of active caspase-8 (lanes 9–12) at 371C for 60 min. Lanes 1–4 show the ‘input’ or corresponding labeled translation products that went into each reaction. Cleavage reactions were resolved by SDS-PAGE, and the gels fixed, dried and autoradiographed

procaspase-3.5,7 DISC assembly in type II cells, on the other components of different apoptotic pathways among the two hand, is both delayed and subdued and the resulting species, it was important to investigate further the biochemical autoprocessed caspase-8 insufficient for the direct activation basis for this difference in responsiveness. of caspase-3. Caspase-8 can still cleave the Bcl-2 family Apoptosis is discernible by flow cytometry within 2 h of protein, Bid, however, to generate a truncated active form of addition of TNF-a/CHX, even as overexpressed Bcl-xL is able the latter that translocates to the mitochondria and promotes to prevent mitochondrial cyt c release for at least 4 h in FL5.12 the release of mitochondrial cyt c into the cytosol.8-10 Most cells. However, while Johnson et al.15 observed no cyt c activated effector caspases have the ability to cleave other release for 6 h in response to TNF-a/CHX, our studies show caspases, including initiator caspases, but such processing that Bcl-xL FL5.12 cells release almost 50% of their cyt c into activity is unregulated and primarily associated with the the cytoplasm by the end of 6 h under the same conditions degradative phase of the apoptotic process. Caspase-2, -6 (Figure 2b). The FL5.12 Bcl-xL lines used in the present study and -9, for instance, are efficiently processed by activated have been selected for high-level expression of transfected 22 caspase-3 during the rapid cell death observed in type I cells Bcl-xL and have consistently exhibited this delayed release in response to Fas ligand.24,29 In these cells, caspase-9 of cyt c in response to TNF-a, while protecting mitochondria functions not as an initiator, but as one more target of an for over 72 h following growth factor (IL-3) withdrawal. We effector caspase. Caspase-9 activation follows that of believe that the positive feedback of the cytosolic caspase caspase-3 in such a pathway. Survival members of the Bcl- activity on the mitochondria causes the release of cyt c as a 2 family, by virtue of their ability to prevent cyt c release, late event during the apoptotic process.30 This feedback loop therefore, impart almost total protection against apoptosis could be manifest in a number of ways, including cleavage of induced via Fas receptor crosslinking to type II cells. The the membrane-inserted survival-promoting proteins them- murine FL5.12 cell line in the present study exhibits both type I selves.31,32 and type II characteristics. Preventing cyt c release by We show that caspase-9 is processed within 2 h of overexpressing an antiapoptotic Bcl-2 family protein, does activation of the death receptor pathway in Bcl-xL-expressing not protect FL5.12 cells from death receptor activation- FL5.12 cells. Additionally, using specific inhibitors we observe induced apoptosis15 (and Figure 1). Given the close con- that caspase-9 activity is essential for the early amplification of servation of specific death pathways among humans and apoptosis, under these circumstances. This processing of mice, as well as the high degree of homology shared by caspase-9 proceeds in the absence of mitochondrial cyt c

Cell Death and Differentiation Activation of caspase-9 by caspase-8 MA McDonnell et al 1012

release and occurs too early to be the result of a feedback loop that DQLD (D330), rather than PEPD (D315), is the preferred involving active caspase-3.24 Furthermore, the Western blots site of cleavage activity. This difference in susceptibility in Figure 3 (lower panels) show relatively low amounts of between hC-9 and mC-9 to processing by active caspase-8 cleaved/active caspase-3 resulting from caspase-8 proces- may help explain the difference in response to death receptor sing activity at 2 and 3 h as compared to the cleaved form activation between human cells of type I and murine FL5.12 detected at later time points. Additionally, the first detectable cells. Additional evidence that hC-9 and mC-9 are differently processing intermediate is the 38 kDa product, and not the regulated comes from a study showing that the Akt 40 kDa band expected from caspase-3 cleavage activity. The phosphorylation site (located at Serine196) presumed to be processing of procaspase-9 by active caspase-3 (at residue involved in the inactivation of hC-9 is absent from its murine D368), known to occur during amplification of caspase counterpart.37,38 cascades, generates a 40 kDa cleavage product in the in In mitochondrial pathways of apoptosis, the zymogen form vitro cleavage assays (shown in Figure 8b), and confirms this. of caspase-9 is rapidly recruited into the apoptosome The inhibitor studies further confirm that active caspase-3 is following the release of cyt c.39 The active form of caspase- not the protease responsible for the observed early caspase-9 9 is believed to be the Apaf-1-bound holoenzyme. Since processing (Figures 4b and 5b). Procaspase-9 has been procaspase-9 is not normally detected in the apoptosome, it is shown to be cleaved as cells undergo apoptosis; however, thought to undergo rapid autoprocessing. However, studies such cleavage occurs late during apoptotic progression and is with noncleavable mutants have shown that the unprocessed thought to be involved in the turnover of the protease rather caspase can also associate with oligomerized Apaf-1 and than in its enzymatic activation.17,33 rapidly recruit and activate caspase-3.16,17 It is unlikely that a Apaf-1 oligomerization in the presence of cyt c and dATP holoenzyme is involved in the processing and activation of has been shown to be the primary cause of the autoproces- caspase-9 in response to TNF-a in the FL5.12 cells. Our data sing that results in the activation of caspase-9.12,23,34. suggest, instead, that caspase-9 is functioning not as an Although the full-length Apaf-1 molecule requires cyt c for initiator, but as an effector caspase and as a direct substrate oligomerization, a truncated form of Apaf-1 lacking the WD-40 for active caspase-8 early after apoptotic stimulation. The repeat region has been shown to be constitutively active in possibility that the murine caspase-9 cleavage site is modified vitro.18 We were able to detect truncated forms of Apaf-1 in in healthy, proliferating cells in a manner similar to the Bid vivo in response to TNF-a/CHX in the FL5.12 cells (not protein is an attractive one and currently under investigation. shown), but these truncated forms appeared 3–4 h after the We propose the following model to explain our observa- initiation of the signal, too late to initiate caspase-9 autopro- tions. In addition to cleaving the Bcl-2 protein Bid and small cessing and were more likely the result of caspase-3 amounts of procaspase-3, active caspase-8 molecules in processing activity.35,36 Furthermore, we could demonstrate FL5.12 cells cleave and activate procaspase-9 in response that caspase-9 cleavage occurs within 3 h of TNF-receptor to TNF-receptor crosslinking (see Figure 9). The cleaved Bid occupation in Apaf-1-deficient MEFs (Figure 7). (tBid) promotes the release of cyt c, causing rapid amplifica- Caspase activity determinations in both FL5.12 Bcl-xL cells tion of apoptosis via apoptosome formation and caspase-9 and Apaf-1À/À MEFs, using colorimetric substrates, demon- activation.8-10 In the absence of cyt c release, however, strated that the processed caspase-9 protein had caspase caspase-9 that has been processed directly by caspase-8 activity (Figure 5b, and not shown). Additionally, FL5.12 Bcl-xL cleaves and activates caspase-3, which, in turn, processes cells expressing half as much caspase-9 protein as the more caspase-9 in a positive feedback amplification loop. It parental controls, showed 30% increased viability when may be argued that a cell overexpressing Bcl-xL is not a exposed to TNF-a/CHX for 6 h (Figure 6). The partial ‘normal’ cell and cannot, therefore, serve as a model for protection imparted by downregulated caspase-9 is strong understanding mechanisms of apoptosis. We believe we have indication that the activity of this caspase contributes to the identified a novel pathway of amplification of the caspase early, rapid apoptosis observed in the parental cells (Figure 1b). The 38 kDa processing intermediate of caspase-9 appears to be a product of caspase-8 cleavage activity. In vitro TNF receptor occupation cleavage assays, using active recombinant caspase-8, cell surface confirm that the 38 kDa band is a major processing product Bcl-xL and is a result of cleavage at the SEPD (D353) and not the DISC assembly Bid cleavage/ Caspase-8 activation translocation LDSD motif (D349) on murine caspase-9 (Figure 6). Further- more, the early appearance of the shorter processing weak mitochondrial damage cyto c/Smac/AIF release intermediate, in TNF-treated FL5.12 cells, supports the possibility that caspase-9 is a direct processing substrate for Caspase-3 and other strong effector caspase Caspase-9 cleavage apoptosome active caspase-8 in this pathway of apoptosis. In vitro cleavage/activation and activation assembly cleavage data demonstrate that human caspase-9 is also

cleaved, albeit less efficiently than its murine counterpart, caspase suggesting that the SEPD motif serves as a better substrate cascade for active caspase-8 than does the PEPD site (Figure 6a). DEATH Previously published studies demonstrating the processing of Figure 9 Simplified model of the apoptotic pathways activated following TNF purified recombinant hC-9 by active caspase-828, indicate receptor occupation in murine cells

Cell Death and Differentiation Activation of caspase-9 by caspase-8 MA McDonnell et al 1013 cascade; in order to appreciate the cyt c -independent nature Cyt c release studies of the process, it is important to use a cell line in which cyt c Mitochondrial and cytosolic (S100) fractions were prepared by resuspend- release can be prevented or delayed. Although we have ing 1  107 FL5.12 cells in 0.8 ml ice-cold buffer A (250 mM sucrose, investigated this pathway in a murine pro-B system, similar 20 mM HEPES, 10 mM KCl, 1.5 mM EDTA, 1 mM EGTA, 1 mM DTT, mechanisms are undoubtedly utilized in other murine cell 17 mg/ml aprotinin, 2 mg/ml leupeptin (pH 7.4)). Cells were homogenized types. Apaf-1-deficient fibroblasts offer insight into alternate using a prechilled cylinder cell homogenizer (H&Y Enterprise Redwood pathways since cyt c-induced activation is also effectively shut City, CA, USA), following which the unlysed cells and nuclei were pelleted off in these cells. It is also relevant to note that a number of at 750  g for 25 min. The pellet, representing the mitochondrial fraction cancers resistant to apoptotic induction overexpress anti- 40 was resuspended in buffer A, and the supernatant subjected to further apoptotic Bcl-2 proteins, and activating the apoptotic centrifugation at 100 000  g for 1 h. The supernatant from the final process in such cells by alternative means can be an centrifugation represented the cytosolic fraction. Equivalent amounts of important goal in cancer therapy. mitochondrial and cytosolic (S100) fractions were then Western blotted as A recent study showed that caspase-12 was able to process 20 previously described, first with an antibody against cyt c (gift from R and activate caspase-9 in response to ER stress, while Jemmerson) followed by an antibody against COX IV (Molecular Probes). another indicated that Sendai virus infection could trigger a novel, yet unidentified, pathway of caspase-9 cleavage.19 We have presented evidence that implicates caspase-8 in the Cell viability and caspase activity assays processing and activation of caspase-9 in death receptor- Viability of FL5.12 cells was measured at 2 or 3 h intervals over a period activated pathways. All three studies clearly indicate that 24 h following the addition of TNF-a (5 ng/ml) and CHX (20 mg/ml) to the caspase-9 can also be activated by mechanisms involving medium, or at 24 h intervals for 4 or 5 days following withdrawal of IL-3 neither cyt c release nor Apaf-1 activation. In this capacity, the from the medium.41 For the caspase inhibitor studies, FL5.12 cells were activated protease probably contributes to the amplification of incubated with z-DQMD-fmk, z-IETD-fmk or z-LEHD-fmk at 100 mM death pathways that have already been initiated. A cell concentration or z-VAD-fmk (50 mM) for 30 min prior to the addition of programmed to die utilizes all the means within its control to TNF-a and CHX. For the IL-3 studies, cells were washed three times in die a quick death. It stands to reason, therefore, that proteins, growth medium lacking IL-3 and resuspended in this medium at a such as caspase-9, functioning at critical points of apoptotic concentration of 5 105 cells/ml. Aliquots were withdrawn at specified pathways have the ability to amplify the apoptotic process by  time intervals, and viability determined by flow cytometry using PI more than just one mechanism to ensure the cell’s early and exclusion alone or a combination of Annexin V–FITC uptake and PI unobtrusive demise. exclusion using protocol suggested by BioVision, Inc. DEVDase, IETDase and LEHDase activities in cell lysates (from 2  106 cells per data point) Materials and Methods were determined using colorimetric assay kits (R&D Systems) according to the manufacturer’s protocol. Cell lines, antibodies and plasmid constructs 41 FL5.12 murine pro-B lymphoma cells were cultured as described earlier. Western blotting For plasmid transfections, 1  107 FL5.12 cells were electroporated at FL5.12 cells were pelleted, washed in phosphate-buffered saline, and then 960 mF and 250 V with 10 mg of plasmids, pSFFV-Neo or pSFFV-Bcl-xL, described previously.42 Neomycin-resistant cells were selected in medium lysed in RIPA buffer containing 1% Nonidet P-40, 1% deoxycholate and containing 1 mg/ml G418. Single cell clones were obtained from 0.1% SDS, supplemented with protease inhibitor cocktail (Calbiochem). transfectant pools by limiting dilution cloning in 96-well microtiter plates. Adherent MEFs were detached from culture plates with Accutase Apaf-1 knockout and control primary embryo fibroblasts (a gift from Scott (Innovative Cell Technologies, Inc.) and pelleted, together with Lowe, Cold Spring Harbor) were grown in Dulbecco’s modified Eagle’s predetached floaters, as described above. The lysed cells were medium supplemented with 10% fetal calf serum, 2 mM glutamine, 100 U/ centrifuged at 14 000  g to remove cellular debris. Protein concentrations ml penicillin, 100 mg/ml streptomycin, and 10 mM b-mercaptoethanol. of extracts were determined by the colorimetric bicinchoninic acid method Active recombinant caspase-3 was from Pharmingen and active caspase- (Pierce Chemical Company). Equal amounts of protein were electro- 8 from BioVision, Inc. phoretically separated in 14% SDS-PAGE and transferred to nitrocellu- Caspase-9 monoclonal antibody, cleaved caspase-3 polyclonal anti- lose. Membrane blocking, washing, primary and secondary antibody body and the Apaf-1 monoclonal antibody were purchased from Stressgen incubations and chemiluminescence reactions were carried out according Biotechnologies, Cell Signaling Technology, and Chemicon International, to the Amersham ECL protocol. Antibody dilutions were carried out as respectively. The cyt c antibody was a gift from R Jemmerson (University suggested on the data sheet provided by the manufacturing company. of Minnesota, USA). The COX IV antibody was purchased from Molecular Blots were stripped for reuse by washing for 30 min to 2 h in TBS-T buffer Probes and the actin antibody was obtained from Oncogene. The hC-9 (pH 3.0) at room temperature. construct was a gift from E Alnemri (Thomas Jefferson University). Full- length mC-9 cDNA was synthesized from mouse brain RNA by PCR using specific oligos, and cloned into the BamHI and XhoI sites of the pCDNA siRNA construction and transfection 3.0 vector (Invitrogen). The myc-tagged construct, mC-9myc, was siRNAs were generated according to the protocol accompanying the generated by subcloning mC-9 into BamHI and XhoI sites of a pCmyc Silencer siRNA kit (Ambion, Inc.). Briefly, sense and antisense oligos were vector (gift from G Nunez, University of Michigan, USA). The two point synthesized to target a 21-nucleotide mC-9 sequence (AAGCAGGATC- mutants of mC-9myc (LDSA and SEPA) converting D349 or D353, CAGAGGCTGTT-30), with an additional 8-nucleotide leader sequence respectively, to alanine, were generated by a two-step PCR method. complementary to a T7 promoter primer. Each oligo was hybridized to the

Cell Death and Differentiation Activation of caspase-9 by caspase-8 MA McDonnell et al 1014

T7 promoter primer, filled in with Klenow, and used as a template for 11. Zou H, Henzel WJ, Liu X, Lutschg A and Wang X (1997) Apaf-1, a human transcription by T7 polymerase. Sense and antisense in vitro transcripts protein homologous to C. elegans CED-4, participates in cytochrome c- were hybridized, following which overhanging leader sequences and dependent activation of caspase-3 (see comments). Cell 90: 405–413 residual DNA templates were removed by treatment with RNase and 12. Zou H, Li Y, Liu X and Wang X (1999) An APAF-1 cytochrome c multimeric complex is a functional apoptosome that activates procaspase-9. J. Biol. Chem. Dnase, respectively. Finally, the double-stranded RNAs were purified by 274: 11549–11556 passing through a filter cartridge and eluted in nuclease-free water. FL5.12 13. Susin SA, Zamzami N, Castedo M, Hirsch T, Marchetti P, Macho A, Daugas E, cells were either mock-transfected or transfected with the siRNA (100 nM) Geuskens M and Kroemer G (1996) Bcl-2 inhibits the mitochondrial release of in the presence of transfection agent siPORT Amine (Ambion, Inc.) for 6 h. an apoptogenic protease. J. Exp. Med. 184: 1331–1341 A fraction of the transfected population was lysed and immunoblotted with 14. Yin XM, Wang K, Gross A, Zhao Y, Zinkel S, Klocke B, Roth KA and Korsmeyer caspase-9 antibodies, and the remaining cells were used for the viability SJ (1999) Bid-deficient mice are resistant to Fas-induced hepatocellular apoptosis. Nature 400: 886–891 studies described above. 15. Johnson BW, Cepero E and Boise LH (2000) Bcl-xL inhibits cytochrome c release but not mitochondrial depolarization during the activation of multiple death pathways by tumor necrosis factor-a. J. Biol. Chem. 275: In vitro caspase cleavage assays 31546–31553 16. Bratton SB, Walker G, Srinivasula SM, Sun XM, Butterworth M, Alnemri ES and Radiolabeled mC-9-myc protein, LDSA, or SEPA proteins were Cohen GM (2001) Recruitment, activation and retention of caspases-9 and -3 synthesized in vitro using the TNT transcription/translation system by Apaf-1 apoptosome and associated XIAP complexes. EMBO J. 20: 998– 35 (Promega) and 20 mCi [ S]methionine (Amersham/Pharmacia) per 1009 reaction. Active human recombinant caspase-3 (15 mU/ml), or -8 17. Stennicke HR, Deveraux QL, Humke EW, Reed JC, Dixit VM and Salvesen GS (150 mU/ml), was used to cleave the in vitro translated proteins at 371C (1999) Caspase-9 can be activated without proteolytic processing. J. Biol. for 60 min using 5 ml in vitro translation mix as substrate in a 25 ml total Chem. 274: 8359–8362 reaction volume. Reactions were stopped with an equal volume of 2 Â 18. Adrain C, Slee EA, Harte MT and Martin SJ (1999) Regulation of apoptotic protease activating factor-1 oligomerization and apoptosis by the WD-40 repeat Laemmli sample buffer containing reducing agent. The resulting cleavage region. J. Biol. Chem. 274: 20855–20860 products were separated by SDS-PAGE, and the gels were fixed, dried 19. Bitzer M, Armeanu S, Prinz F, Ungerechts G, Wybranietz W, Spiegel M, and autoradiographed. Bernlohr C, Cecconi F, Gregor M, Neubert WJ, Schulze-Osthoff K and Lauer UM (2002) Caspase-8 and Apaf-1-independent caspase-9 activation in Sendai virus- infected cells. J. Biol. Chem. 277: 29817–29824 20. Morishima N, Nakanishi K, Takenouchi H, Shibata T and Yasuhiko Y (2002) An Acknowledgements endoplasmic reticulum stress-specific caspase cascade in apoptosis. The authors are grateful to Craig Thompson for stimulating discussions Cytochrome c-independent activation of caspase-9 by caspase-12. J. Biol. Chem. 277: 34287–34294 and for a critical reading of the manuscript. We also thank Manuel 21. Vander Heiden MG, Chandel NS, Williamson EK, Schumacker PT and Melendez for excellent technical assistance. This work was supported by a Thompson CB (1997) Bcl-xL regulates the membrane potential and volume grant from the Leukemia Research Fund. homeostasis of mitochondria (see comments). Cell 91: 627–637 22. Rathmell JC, Vander Heiden MG, Harris MH, Frauwirth KA and Thompson CB (2000) In the absence of extrinsic signals, nutrient utilization by lymphocytes is insufficient to maintain either cell size or viability. Mol. Cell 6: 683–692 References 23. Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES and Wang X (1997) Cytochrome c and dATP-dependent formation of 1. Ellis RE, Yuan JY and Horvitz HR (1991) Mechanisms and functions of cell Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 91: death. Annu. Rev. Cell Biol. 7: 663–698 479–489 2. Wyllie AH, Kerr JF and Currie AR (1980) Cell death: the significance of 24. Slee EA, Adrain C and Martin SJ (1999) Serial killers: ordering caspase apoptosis. Int. Rev. Cytol. 68: 251–306 activation events in apoptosis. Cell Death Differ. 6: 1067–1074 3. Thornberry NA and Lazebnik Y (1998) Caspases: enemies within. Science 281: 25. Slee EA, Harte MT, Kluck RM, Wolf BB, Casiano CA, Newmeyer DD, Wang 1312–1316 HG, Reed JC, Nicholson DW, Alnemri ES, Green DR and Martin SJ (1999) 4. Budihardjo I, Oliver H, Lutter M, Luo X and Wang X (1999) Biochemical Ordering the cytochrome c-initiated caspase cascade: hierarchical activation of pathways of caspase activation during apoptosis. Annu. Rev. Cell Dev. Biol. 15: caspases-2, -3, -6, -7, -8, and -10 in a caspase-9-dependent manner. J. Cell 269–290 Biol. 144: 281–292 5. Scaffidi C, Fulda S, Srinivasan A, Friesen C, Li F, Tomaselli KJ, Debatin KM, 26. Nicholson DW (1999) Caspase structure, proteolytic substrates, and function Krammer PH and Peter ME (1998) Two CD95 (APO-1/Fas) signaling during apoptotic cell death. Cell Death Differ. 6: 1028–1042 pathways. EMBO J. 17: 1675–1687 27. Kuida K (2000) Caspase-9. Int. J. Biochem. Cell Biol. 32: 121–124 6. Medema JP, Scaffidi C, Kischkel FC, Shevchenko A, Mann M, Krammer PH 28. Srinivasula SM, Ahmad M, Fernandes-Alnemri T, Litwack G and Alnemri and Peter ME (1997) FLICE is activated by association with the CD95 death- ES (1996) Molecular ordering of the Fas-apoptotic pathway: the Fas/ inducing signaling complex (DISC). EMBO J. 16: 2794–2804 APO-1 protease Mch5 is a CrmA-inhibitable protease that activates 7. Muzio M, Stockwell BR, Stennicke HR, Salvesen GS and Dixit VM (1998) An multiple Ced-3/ICE- like cysteine proteases. Proc. Natl. Acad. Sci. USA 93: induced proximity model for caspase-8 activation. J. Biol. Chem. 273: 2926– 14486–14491 2930 29. Hirata H, Takahashi A, Kobayashi S, Yonehara S, Sawai H, Okazaki T, 8. Li H, Zhu H, Xu CJ and Yuan J (1998) Cleavage of BID by caspase 8 mediates Yamamoto K and Sasada M (1998) Caspases are activated in a branched the mitochondrial damage in the Fas pathway of apoptosis. Cell 94: 491–501 protease cascade and control distinct downstream processes in Fas-induced 9. Luo X, Budihardjo I, Zou H, Slaughter C and Wang X (1998) Bid, a Bcl2 apoptosis. J. Exp. Med. 187: 587–600 interacting protein, mediates cytochrome c release from mitochondria in 30. Bossy-Wetzel E and Green DR (1999) Caspases induce cytochrome c release response to activation of cell surface death receptors. Cell 94: 481–490 from mitochondria by activating cytosolic factors. J. Biol. Chem. 274: 17484– 10. Wei MC, Lindsten T, Mootha VK, Weiler S, Gross A, Ashiya M, Thompson CB 17490 and Korsmeyer SJ (2000) tBID, a membrane-targeted death ligand, 31. Cheng EH, Kirsch DG, Clem RJ, Ravi R, Kastan MB, Bedi A, Ueno K and oligomerizes BAK to release cytochrome c (in process citation). Genes Dev. Hardwick JM (1997) Conversion of Bcl-2 to a Bax-like death effector by 14: 2060–2071 caspases. Science 278: 1966–1968

Cell Death and Differentiation Activation of caspase-9 by caspase-8 MA McDonnell et al 1015

32. Clem RJ, Cheng EH, Karp CL, Kirsch DG, Ueno K, Takahashi A, Kastan MB, 37. Cardone MH, Roy N, Stennicke HR, Salvesen GS, Franke TF, Stanbridge E, Griffin DE, Earnshaw WC, Veliuona MA and Hardwick JM (1998) Modulation of Frisch S and Reed JC (1998) Regulation of cell death protease caspase-9 by cell death by Bcl-XL through caspase interaction. Proc. Natl. Acad. Sci. USA phosphorylation (see comments). Science 282: 1318–1321 95: 554–559 38. Fujita E, Jinbo A, Matuzaki H, Konishi H, Kikkawa U and Momoi T (1999) Akt 33. Rodriguez J and Lazebnik Y (1999) Caspase-9 and APAF-1 form an active phosphorylation site found in human caspase-9 is absent in mouse caspase-9. holoenzyme. Genes Dev. 13: 3179–3184 Biochem. Biophys. Res. Commun. 264: 550–555 34. Srinivasula SM, Ahmad M, Fernandes-Alnemri T and Alnemri ES (1998) 39. Cain K, Bratton SB and Cohen GM (2002) The Apaf-1 apoptosome: a large Autoactivation of procaspase-9 by Apaf-1-mediated oligomerization. Mol. Cell caspase-activating complex. Biochimie. 84: 203–214 40. Strasser A, O’Connor L, Huang DC, O’Reilly LA, Stanley ML, Bath ML, Adams 1: 949–957 JM, Cory S and Harris AW (1996) Lessons from bcl-2 transgenic mice for 35. Bratton SB, Walker G, Roberts DL, Cain K and Cohen GM (2001) Caspase-3 immunology, cancer biology and cell death research. Behring. Inst. Mitt. cleaves Apaf-1 into an approximately 30 kDa fragment that associates with an 101–117 inappropriately oligomerized and biologically inactive approximately 1.4 MDa 41. Boise LH, Gonzalez-Garcia M, Postema CE, Ding L, Lindsten T, Turka LA, Mao apoptosome complex. Cell Death Differ. 8: 425–433 X, Nunez G and Thompson CB (1993) bcl-x, a bcl-2-related gene that functions 36. Lauber K, Appel HA, Schlosser SF, Gregor M, Schulze-Osthoff K and as a dominant regulator of apoptotic cell death. Cell 74: 597–608 Wesselborg S (2001) The adapter protein apoptotic protease-activating factor- 42. Kelekar A, Chang BS, Harlan JE, Fesik SW and Thompson CB (1997) Bad is a 1 (Apaf-1) is proteolytically processed during apoptosis. J. Biol. Chem. 276: BH3 domain-containing protein that forms an inactivating dimer with Bcl-xL. 29772–29781 Mol. Cell. Biol. 17: 7040–7046

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